A variety of techniques have been developed for preparing directional RNA libraries for sequencing. For example, some methods designed to amplify low quantities of RNA involve the use of “template switching.” A single-stranded cDNA library is synthesized from RNA template by hybridization and extension of a primer comprised of a 3′-terminal degenerate sequence or poly-dT sequence, which hybridizes to the RNA template, and a non-hybridized 5′ fixed sequence. Upon full-length reverse transcription of the RNA template, cDNA are further extended by the intrinsic 3′ terminal transferase activity of reverse transcriptase, typically incorporating a short poly-dC sequence. A second oligonucleotide sequence comprised of the identical 5′ fixed sequence as in the cDNA primer and a 3′ sequence (typically poly G), terminally blocked to prevent extension, then is hybridized to the terminal transferase-extended poly-dC sequence. This second oligo serves a template for continued extension of the cDNA strand, allowing for affixing of additional sequence that is complementary to the 5′ fixed sequence. Following a purification step, a single amplification primer comprised of the 5′ fixed sequence is introduced to the cDNA library. The cDNA library is amplified by PCR. This method requires about 100 pg to about 100 ng of rRNA-depleted RNA.
Other RNA amplification methods employ ligation, which reduces amplification efficiency by at least 10-fold. These methods typically require two or more solid phase reversible immobilized (SPRI) paramagnetic bead purification steps. As an example, one method comprises performing first-strand cDNA synthesis with a primer mixture of random degenerate sequence and poly-dT, followed by second-strand synthesis in the presence of dUTP. The resulting double-stranded cDNA library is fragmented and concentrated by SPRI paramagnetic bead purification. Following end repair and A-tailing, sequencing adaptors are ligated to the double-stranded cDNA, and these constructs are digested with uracil DNA glycosylase, eliminating the second cDNA strand. The first strand, with affixed adapter sequences, is purified a second time on SPRI beads, and PCR-amplified. This method requires from about 10 ng to about 100 ng of total RNA.
There is a need, therefore, for amplification methods that are amenable to low RNA input quantities and/or low quality RNA, devoid of purification steps, and clearly indicate amplification product strandedness.